The Role of Dimerization in Raf Signaling

Normal growth factor-induced signaling is dependent upon the Rafs dimerizing at the plasma membrane in a Ras-dependent manner. The Rafs then become activated and phosphorylate their downstream target, MEK. Signaling via an oncogenic RTK or Ras, or a moderate, low, or impaired activity Raf mutant is also dependent upon Raf dimerization. The DI1 peptide, which mimics part of the B-Raf dimer interface, can inhibit this disease-associated signaling by blocking Raf dimerization.

Normal growth factor-induced signaling is dependent upon the Rafs dimerizing at the plasma membrane in a Ras-dependent manner. The Rafs then become activated and phosphorylate their downstream target, MEK. Signaling via an oncogenic RTK or Ras, or a moderate, low, or impaired activity Raf mutant is also dependent upon Raf dimerization. The DI1 peptide, which mimics part of the B-Raf dimer interface, can inhibit this disease-associated signaling by blocking Raf dimerization.

One frequently mutated pathway in a variety of cancers and developmental disorders is the Ras-Raf-MEK-ERK cascade. Normally, binding of a growth factor to its receptor switches on Ras, which, in turn, activates one or more of the Raf kinase family members, A-Raf, B-Raf, and C-Raf. Rafs perpetuate the signal by phosphorylating and activating MEK, another kinase that phosphorylates a third kinase, ERK. ERK then phosphorylates a number of key growth-, survival-, or differentiation-promoting targets. Of the proteins in the cascade, Rafs have the most complex regulatory mechanisms, including the ability to form dimers. Because the role that dimerization plays in Raf function has been unclear, researchers working with Deborah Morrison, Ph.D., Chief of CCR’s Laboratory of Cell and Developmental Signaling, decided to investigate its significance in normal and disease-associated Raf signaling.

The scientists began their studies by asking whether all three Raf proteins were capable of forming dimers. Using an antibody technique called co-immunoprecipitation, they harvested each Raf protein from serum-starved or epidermal growth factor (EGF)-treated cells and examined the associated proteins. Following EGF stimulation, the researchers observed a strong interaction between B-Raf and C-Raf. In contrast, A-Raf only weakly bound B-Raf and failed to bind C-Raf.

To determine whether these binding abilities affect Raf function, the investigators used small hairpin RNAs to knock-down each Raf isoform and then determined the kinase activities of the remaining Raf proteins. In cells lacking A-Raf, there was no difference in B-Raf or C-Raf activity following EGF treatment. With the loss of B-Raf, however, the activation of both A-Raf and C-Raf in response to EGF decreased. In fact, C-Raf retained only 10 percent of its normal activity. In C-Raf-depleted cells, EGF-stimulated A-Raf activity was unchanged while B-Raf activity was halved. These results suggest that the ability of Rafs to form dimers does affect their kinase activities. Because A-Raf exhibited weak dimerization and only a small change in activity with the loss of B-Raf, the researchers focused the remainder of their studies on B- and C-Raf.

Previous studies of the structures of Raf proteins had identified amino acids key to dimerization. The investigators decided to mutate two of these residues to see how they affect the formation of homodimers (composed of two C-Raf or B-Raf proteins) and heterodimers (composed of one B-Raf and one C-Raf protein) as well as B- and C-Raf activation. The first mutation, R>H, in either B- or C-Raf did not affect binding to a number of signaling proteins, including Ras, but did block the formation of both homodimers and heterodimers. This mutation also blunted B- and C-Raf enzymatic activities in response to EGF. In contrast, the E>K mutation enhanced dimerization as well as kinase activation. C-Raf homodimers were especially enhanced by this mutation. Interestingly, the researchers observed B-Raf homodimers even in the absence of EGF for normal and E>K B-Raf.

The researchers next asked whether dimerization was important for the function of a collection of disease-associated Raf mutants with a spectrum of kinase activities. They made R>H and E>K mutations to six oncogenic B-Raf mutants and two C-Raf mutants associated with Noonan syndrome. As with the normal Rafs, the R>H mutation prevented heterodimer formation while the E>K mutation enhanced it in all eight disease-associated mutants.

To evaluate the biological effect of dimerization on these mutants, the investigators measured the ability of cells expressing each mutant to form foci, a measure of cancer potential. Cells expressing mutants with high intrinsic kinase activity formed foci even in the presence of the R>H mutation, indicating that dimerization is not required for their activity. Cells that expressed Raf mutants with weaker activity, however, failed to form foci with the R>H mutation. In contrast, the E>K mutation increased foci formation induced by Raf mutants with low or impaired kinase activity, suggesting that dimerization is critical for their activation.

Since dimer formation affects the biological activity of Raf proteins, blocking Raf dimerization may have therapeutic potential. The researchers tested this idea by generating a series of peptides to interfere with Raf dimerization. One peptide, DI1, strongly bound to both B-Raf and C-Raf and blocked normal Raf-mediated activation of MEK in response to EGF. Likewise, DI1 prevented B- and C-Raf heterodimerization. In agreement with data from the focus formation assay, DI1 could block MEK activation by the disease-associated Raf mutants except for those with high intrinsic activity like V600E-B-Raf. Treating cancer cells that expressed Ras or B-Raf mutations, other than V600E, with DI1 also prevented their proliferation and decreased their viability.

These data confirm the importance of dimerization in the regulation of Raf activity and suggest that targeting Raf dimer interface may be an effective treatment for Raf-associated diseases, including some cancers.

Summary Posted: 02/2013

Reference

Freeman AK, Ritt DA, and Morrison DK. Effects of Raf Dimerization and Its Inhibition on Normal and Disease-Associated Raf Signaling. Mol Cell. January 22, 2013. PubMed Link